Intranasal vaccination with lipid-conjugated immunogens promotes antigen transmucosal uptake to drive mucosal and systemic immunity

To combat the HIV epidemic and emerging threats such as SARS-CoV-2, immunization strategies are needed that elicit protection at mucosal portals of pathogen entry. Immunization directly through airway surfaces is effective in driving mucosal immunity, but poor vaccine uptake across the mucus and epithelial lining is a limitation. The major blood protein albumin is constitutively transcytosed bidirectionally across the airway epithelium through interactions with neonatal Fc receptors (FcRn). Exploiting this biology, here, we demonstrate a strategy of “albumin hitchhiking” to promote mucosal immunity using an intranasal vaccine consisting of protein immunogens modified with an amphiphilic albumin-binding polymer-lipid tail, forming amph-proteins. Amph-proteins persisted in the nasal mucosa of mice and nonhuman primates and exhibited increased uptake into the tissue in an FcRn-dependent manner, leading to enhanced germinal center responses in nasal-associated lymphoid tissue. Intranasal immunization with amph-conjugated HIV Env gp120 or SARS-CoV-2 receptor binding domain (RBD) proteins elicited 100- to 1000-fold higher antigen-specific IgG and IgA titers in the serum, upper and lower respiratory mucosa, and distal genitourinary mucosae of mice compared to unmodified protein. Amph-RBD immunization induced high titers of SARS-CoV-2–neutralizing antibodies in serum, nasal washes, and bronchoalveolar lavage. Furthermore, intranasal amph-protein immunization in rhesus macaques elicited 10-fold higher antigen-specific IgG and IgA responses in the serum and nasal mucosa compared to unmodified protein, supporting the translational potential of this approach. These results suggest that using amph-protein vaccines to deliver antigen across mucosal epithelia is a promising strategy to promote mucosal immunity against HIV, SARS-CoV-2, and other infectious diseases.

Labeled eOD protein and eOD protein amphiphile conjugate (amph-eOD) were prepared using Alexa Fluor (AF) 647 N-hydroxysuccinimide (NHS) ester (Thermo Fisher Scientific) by reaction of fluorophore with eOD or amph-eOD (≥1mg/ml) in 0.1M sodium bicarbonate buffer for 1 hour at 25°C, per the manufacturer instructions. VRC01 was synthesized as previously described (88); labeled VRC01 was prepared using Pierce NHS-Rhodamine (Thermo Fisher Scientific) by reaction of the fluorophore with human VRC01 (≥1mg/ml) in PBS for 1 hour at 25°C, per the manufacturer instructions. Labeled proteins were purified by centrifugal filtration using 10kDa Amicon spin filters; degree of labeling (DOL) was characterized by UV-Vis spectrophotometry and confirmed to be ≥1.0.

Albumin binding: affinity chromatography
Albumin binding of conjugates was evaluated using albumin-immobilized agarose affinity chromatography as previously described (25). Pierce NHS-activated agarose resin (Thermo Fisher Scientific) was functionalized with albumin by adding 26.4 mg bovine serum albumin (BSA) in 4.4 ml PBS directly to 330 mg agarose, per the manufacturer instructions. The resin reaction was mixed for 1 hour at 25°C followed by 4°C overnight, then quenched with 1M Tris-HCl (pH 8.0) followed by extensive washing with PBS. Next, AF647-labeled eOD or amph-eOD was applied to the albumin-functionalized resin (0.3 µM final concentration in 2 ml column volume) and incubated with end-over-end mixing for 2 hours at 37°C. Eluent was collected following column centrifugation at 1000xg for 2 minutes. The amount of protein or amphiphile conjugate retained in the column was determined by measuring AF647 fluorescence (640/670 nm) of the eluent versus starting sample on a fluorescent plate reader and normalizing by DOL.

IVIS trafficking in murine tissues
In vivo trafficking of AF647-labeled amph-eOD and eOD was evaluated following intranasal administration using In Vivo Imaging System (IVIS) fluorescence imaging (Perkin Elmer). Mice were fed an alfalfa-free diet (AIN-93M, Bio-Serv) for the duration of the study, starting 3 days before immunization, to eliminate background auto-fluorescence in the gut. BALB/c mice were immunized intranasally with 5 µg AF647-amph-eOD or AF647-eOD combined with 5 µg saponin monophosphoryl lipid A (MPLA) nanoparticle (SMNP) adjuvant, and compared to a naïve control. Intranasal immunizations were administered dropwise in 20 µl PBS (10 µl per nare with 30 to 60 second interval between nares) with the mouse anesthetized in the supine position. Postadministration, mice remained anesthetized in the supine position for a minimum of 5 minutes to allow for uptake and prevent drainage. After 24 hours, 48 hours, 72 hours, 7 days, and 11 days post-immunization, the following tissues were excised and AF647 fluorescence (radiant efficiency) was measured by IVIS: nasal cavity (snout minus lower mandible), cervical lymph nodes, intestines, mesenteric lymph nodes, liver, and spleen. The nasal cavity was imaged by removing the head from the mouse body, then removing and discarding the lower mandible from the snout; images were collected of the underside ventral surface of the upper palate.

Histology and fluorescence microscopy of mouse nasal epithelium
Nasal cavity samples from FcRn -/and C57BL/6 mice were processed for histology by FFPE (formalin fixed paraffin embedding) as follows: Samples were fixed in 10% neutral buffered formalin (NBF) for 24 hours at 25°C, then transferred into 70% ethanol for storage at 4°C. Fixed samples were decalcified in 10% EDTA disodium salt dihydrate (Sigma-Aldrich) at pH 7.4 for 10 days at 4°C, changing the EDTA solution every 3 days. Decalcified tissues were embedded in paraffin and sliced into 5µm coronal cross-sections using a microtome, starting 1mm in from the nares and proceeding at 500µm step intervals throughout the nasal cavity to a depth of 7.5mm. Sections located 1.5 to 3mm in from the nares were identified as the main site of vaccine deposition for detailed imaging. Slices were mounted on a glass slide and stained with DAPI using Vectashield HardSet Antifade Mounting Medium with DAPI (Vector Laboratories), then imaged using a Leica SP8 laser scanning confocal microscope with 25X water objective or 63X oil objective. Images were processed in ImageJ.

ELISA for albumin quantification
To assay albumin concentrations in the nasal mucosa, nasal wash was collected from C57BL/6 or FcRn -/mice as described above. Concentration of albumin in the nasal secretions was measured using a commercial mouse albumin ELISA kit (Abcam, cat # ab207620) per the manufacturer's instructions.

Flow cytometry analysis of NALT uptake
BALB/c mice were immunized intranasally with 10µg AF647-eOD or AF647-amph-eOD combined with 5µg SMNP. One and four days later, mice were euthanized and the nasal-associated lymphoid tissue (NALT) was isolated by excising the upper palate (90) and processing to a single cell suspension as follows: The upper palate was enzymatically and mechanically digested in 1 ml RPMI-1640 containing 0.8 mg/ml collagenase/dispase (Roche) and 0.1 mg/ml DNase (Roche) by first cutting into less than 1mm chunks using fine-tipped spring-loaded scissors and then mashing in a 1.5ml biomasher tube (Kimble). After incubating for 15 minutes at 37°C with shaking, supernatant was removed and added to 10ml FACS buffer (PBS+1%BSA) at 4°C; the remaining tissue was subjected to a second round of digestion in 1 ml fresh enzyme mix for an additional 15 minutes at 37°C, then supernatant was removed and again added to cold FACS buffer. This FACS buffer solution was centrifuged at 500xg for 5 minutes to pellet cells; cells were washed once in FACS buffer, passed through a 70 µm filter, and finally centrifuged and resuspended in FACS buffer in a V-bottom plate for antibody staining.

Mouse sample collection
Vaginal mucosal fluid was collected from anesthetized mice by vaginal lavage using 75 µl sterile PBS (3x25 µl instillations, each aspirated three to five times) combined with 5 µl of 25X protease inhibitor (EDTA-free SIGMAFAST Protease Inhibitor Cocktail Tablets, Sigma-Aldrich); fluid was centrifuged at 12,000xg for 10 minutes at 4°C to collect supernatant. Fecal wash was collected from mouse fecal pellets (4 pellets of ~0.75cm each per mouse) combined with 300 µl 1X protease inhibitor; samples were vortexed, incubated for 1 hour at 4°C, vortexed a second time, then centrifuged at 13,000xg for 15 minutes at 4°C to collect supernatant. Saliva wash was collected by dispensing 30 µl sterile PBS between the mouse's cheek and gumline (aspirated three to five times), repeated on both sides, and combined with 10 µl of 2X protease inhibitor. All fluid samples were stored in aliquots at -80°C for future analysis.
Post-euthanasia, BM and FRT tissues were collected to evaluate immune memory and resident plasma cell responses in the vaginal mucosa. FRT was isolated from the vaginal opening to the ovaries, cut into 1 to 3 mm chunks using fine-tipped spring-loaded scissors, and digested in 2 ml/sample of RPMI-1640 containing 2 mg/ml collagenase D (Roche), 0.6 U/ml Dispase II (StemCell Technologies), and 0.2 mg/ml DNase I (Roche) for 30 minutes at 37°C with shaking. Samples were then centrifuged at 500xg for 5 minutes to pellet tissue and cells, supernatant discarded, and resuspended in 2ml fresh digestion media for an additional incubation for 30 minutes at 37°C with shaking. The digestion was quenched by adding an equal volume of RPMI-1640 containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. This solution plus remaining tissue was passed through a 70 µm cell strainer using the plunger end of a 1-ml syringe for additional mechanical digestion, then centrifuged at 500xg for 5 minutes and resuspended in 5 ml ACK lysis buffer for 5 minutes at 4°C to lyse residual red blood cells. An equal volume of cRPMI was added to quench the ACK buffer; samples were then centrifuged at 500xg for 5 minutes and rinsed once with cRPMI, passed through a 70 µm filter a second time, and finally centrifuged and resuspended a final time in cRPMI for counting and further analysis by enzymelinked immunosorbent spot (ELISPOT) assay or flow cytometry.
For RBD studies, nasal wash and bronchoalveolar lavage fluid (BALF) were collected to evaluate resident mucosal antibody responses in the upper and lower respiratory tract. Nasal wash was collected from 2x15 µl instillations of PBS, one in each nare (aspirated three to five times), combined with 10µl of 2X protease inhibitor. BALF was collected from 2x1 ml instillations of sterile PBS in the lungs using a 24G x ¾" catheter through the trachea. Both fluid samples were centrifuged at 12,000xg for 10 min at 4°C to collect supernatant, then stored at -80°C.

ELISA analyses of murine antibody titers
Anti-eOD and anti-RBD IgG and IgA binding titers were measured in mouse serum and mucosal samples (vaginal wash, fecal wash, saliva, nasal wash, and BALF) by ELISA. To capture eODspecific antibodies from immunized mice, MAXIsorp (Thermo Fisher Scientific) 96-well plates were coated directly with eOD antigen at 2 µg/ml in PBS overnight at 4°C. To capture RBDspecific antibodies, Costar Polystyrene High Binding 96-well plates (Corning) were coated directly with RBD antigen at 2 µg/ml in PBS overnight at 4°C. Plates were then blocked with PBS + 2% BSA for 2 hours at 25°C. Mouse serum samples were diluted in block buffer (PBS + 2% BSA) starting at 1:100 or 1:200, and mucosal samples were diluted in block buffer starting at 1:10, followed by 4X serial dilutions. For eOD ELISAs, VRC01 at 5 µg/ml was used as a positive control; for RBD ELISAs, mAb CR3022 or Fc-fusion protein ACE2-Fc at 5µg/ml were used as positive controls. Samples were incubated in plates for 2 hours at 25°C, followed by detection with 1:5000 goat anti-mouse IgG-horseradish peroxidase (HRP, BioRad) or 1:2000 goat anti-mouse IgA-HRP (Invitrogen) in block buffer for 1 hour. Plates were developed using 3,3',5,5'-Tetramethylbenzidine (TMB) substrate for 1 to 20 minutes and stopped with 2N sulfuric acid, and the resulting absorbance (A450/A540) was measured on a plate reader. For all titer analyses, samples directly compared across groups were developed for the same amount of time. Cut-off titers are reported as inverse dilutions giving an HRP absorbance (A450 -A540) of 0.2 (RBD) or 0.1 (eOD) based on background.

ELISPOT analysis of mouse plasma cells
IgG and IgA plasma cells were analyzed in BM and FRT tissue at 35 or greater than 52 weeks post-prime, as indicated, using PVDF-MSIP filter plates (0.45 µm High Protein Binding Immobilon-P Membrane filter plates, Millipore) and Mouse IgG/A ELISpot-BASIC kits (Mabtech). To quantify eOD antigen-specific IgG and IgA plasma cells, filter plates were coated with 10 µg/ml eOD in 100 µl sterile PBS and incubated overnight at 4°C; cells were plated at 500,000 and 250,000 cells per well in 100 µl cRPMI. To quantify total IgG and IgA plasma cells, filter plates were coated with 15 µg/ml anti-IgG (purified goat anti-mouse IgG capture antibody, Mabtech) or anti-IgA (monoclonal antibody MT45A, Mabtech), respectively, in 100 µl sterile PBS and incubated overnight at 4°C; cells were plated at 100,000 and 50,000 cells per well in 100 µl cRPMI. Plates were then incubated for 18 to 20 hours at 37°C; spot detection was carried out per manufacturer instructions, and plates were read on a CTL ImmunoSpot Analyzer.

Mouse parenteral control immunization
To compare intranasal immunization to a parenteral control, BALB/c mice were immunized intranasally or subcutaneously at the scruff of the neck with 5 µg amph-eOD combined with 25 µg cdGMP. Mice were primed on day 0 and boosted on day 42. Blood, vaginal, and fecal samples were collected at regular intervals as described above.

ELISA for anti-PEG antibodies
Antibody responses to PEG included in the amph-protein conjugates was assayed by ELISA. Briefly, MaxiSorp ELISA plates were coated with streptavidin at 1 µg/mL in PBS for 4 hours at 25°C, blocked with PBS + 2% bovine serum albumin (BSA) overnight at 4°C, then washed three times with wash buffer (PBS containing 0.2% Tween20). Biotin-PEG-OH (Creative PEGWorks, cat. #PJK-1946) was added to the plates in blocking buffer (1 µg/mL) and incubated for 2 hours at 25°C. After washing plates three times with wash buffer, mouse serum samples and mouse anti-PEG IgG standard antibody (AffinityImmuno kit cat. #EL-141-PEG-mIGG, starting at 1 µg/ml followed by 2X serial dilutions) were added and incubated for 2 hours prior to washing. Anti-mouse IgG-HRP diluted 1:5000 in blocking buffer was used as a detection antibody. Samples were incubated for 1 hour at 25°C before washing and adding TMB substrate, followed by 2 N H₂SO₄ as a stop solution. Absorbance was measured at 450 nm.

ACE2:RBD binding inhibition assay
Functional antibody inhibition of ACE2:RBD binding was measured in mouse serum and BALF as a preliminary indication of neutralizing antibodies using SARS-CoV-2 Surrogate Virus Neutralization Test Kits (Genscript), per manufacturer instructions. Mouse serum was diluted starting at 1:10 and BALF was diluted 1:2, followed by 4X serial dilutions. Inhibition (IC50) was defined as the sample dilution at which 50% reduction in ACE2:RBD binding was observed relative to a negative control (no inhibition).

Pseudovirus-based SARS-CoV-2 neutralization assay
The SARS-CoV-2 pseudoviruses expressing a luciferase reporter gene were generated in an approach similar to those described previously (91, 92). Briefly, the packaging plasmid psPAX2 (AIDS Resource and Reagent Program), luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene), and spike protein-expressing pcDNA3.1-SARS CoV-2 SΔCT were co-transfected into HEK293T cells by lipofectamine 2000 (Thermo Fisher Scientific). The supernatants containing the pseudotype viruses were collected 48 hours post-transfection, which were purified by centrifugation and filtration with 0.45 µm filter. To determine the neutralization activity of mouse serum and mucosal samples, HEK293T expressing human ACE2 (hACE2) were seeded in 96well tissue culture plates at a density of 1.75 x 10 4 cells per well overnight. Samples (serum, saliva, nasal wash, vaginal wash, fecal wash, and BALF) were first heat-inactivated at 56°C for 30 minutes. Three-fold serial dilutions of heat-inactivated serum or mucosal samples were then prepared and mixed with 50 µL of pseudovirus. The mixture was incubated at 37°C for 1 hour before adding to HEK293T-hACE2 cells. At 48 hours after infection, cells were lysed in Steady-Glo Luciferase Assay (Promega) according to the manufacturer's instructions. SARS-CoV-2 neutralization titers (NT50) were defined as the sample dilution at which a 50% reduction in relative light unit (RLU) was observed relative to the average of the virus control wells.

Non-human primates
Six female Indian rhesus macaques (Macaca mulatta) were assigned to the IVIS trafficking study (n = 3 animals per group). Twelve female Indian rhesus macaques between 3 and 4 years of age were assigned to the longitudinal immunization study (n = 6 animals per group). Macaques were distributed such that age, weight, and MHC genotyping were equivalent across groups. Animals were housed and maintained at the New Iberia Research Center (NIRC) of the University of Louisiana at Lafayette in accordance with the rules and regulations of the "Guide for the Care and Use of Laboratory Animals". The entire study (protocol 8789-08) was reviewed and approved by the University of Louisiana at Lafayette IACUC. All animals were negative for SIV, simian T cell leukemia virus and simian retrovirus. The animals were also typed for MHC and those expressing the MamuB*008 or B*017 alleles were excluded whereas those expressing the MamuA*001 allele were distributed equally among the groups.

IVIS trafficking in non-human primate tissues
In vivo trafficking of AF647-labeled amph-eOD and eOD was evaluated following intranasal administration using an IVIS fluorescence imaging system (Perkin Elmer). Macaques were immunized intranasally in a dropwise manner directly to each nostril, 200 µl per nare (400 µl total per animal), with 100 µg AF647-amph-eOD or AF647-eOD mixed with 375 µg SMNP. Postadministration, animals remained in the supine position under anesthesia for 10 minutes to allow for vaccine uptake and to prevent drainage. After 24 hours, the tonsils, adenoids, cervical lymph nodes, axillary lymph nodes, and nasal tissue including turbinates were collected, fixed in 4% paraformaldehyde for 5 days, then transferred to PBS + 0.1% paraformaldehyde + 0.05% sodium azide for storage at 4°C prior to evaluation by IVIS.

ELISA analysis of non-human primate antibody titers
To measure eOD-specific antibody titers, MAXIsorp 96-well plates (Thermo Fisher Scientific) were coated with 2 µg/mL of gp120 eOD monomer in PBS. Serum samples were diluted 1:50 and mucosal washes were diluted 1:10 in 2% BSA block buffer, followed by 4X serial dilutions. hVRC01 at 5µg/ml was included as a positive control. Samples were incubated for 2 hours at room temperature, followed by detection with 1:5000 goat anti-human IgG-HRP (Jackson ImmunoResearch) or 1:2000 goat-anti-human IgA-HRP (Thermo Fisher Scientific). Cut-off titers are reported as inverse dilutions giving an HRP absorbance (A450 -A540) of 0.2 (IgA) or 0.1 (IgG) based on background.

ELISPOT analysis of non-human primate plasma cells
Total and antigen-specific plasmablast responses in peripheral blood were determined by ELISPOT assay as previously described (94). Briefly, 96-well multiscreen HTS filter plates (Millipore) were coated overnight at 4°C with 100 μl/well of 5 μg/ml of goat anti-monkey IgG, IgM or IgA antibodies (Rockland) or of 1 μg/ml of HIV eOD-gp120, respectively. Plates were washed with PBS plus 0.05% Tween 20 (PBS-T) and blocked with complete medium at 37°C for 2 hours. Freshly isolated cells were plated in duplicates in three-fold serial dilutions and incubated overnight in a 5% CO2 incubator at 37°C. Plates were washed with PBS-T and incubated with biotin-conjugated anti-monkey IgG, IgM or IgA antibodies (Rockland) diluted 1:1000 for 1 hour at 37°C. After washing, plates were incubated with HRP-conjugated streptavidin diluted 1:1,000 (Vector Labs) at 25°C for 2 hours and developed using the AEC substrate kit (BD Biosciences). To stop the reaction, plates were washed extensively with water followed by air drying. Spots were imaged and counted using an Immunospot ELISPOT Analyzer (Cellular Technology Limited); the number of spots specific for each Ig isotype was reported as the number of either total or antigen-specific antibody-producing cells per million PBMCs.    Fig. 4A and F with saponin (collected at week 11) or cdGMP adjuvants (collected at week 12) were analyzed by ELISA for anti-PEG IgG, comparing to a reference anti-PEG IgG standard.  Figure S10. Intranasal immunization with amph-protein conjugates leads to improved humoral immune responses in non-human primates. Rhesus macaques (n = 6 female animals per group) were immunized i.n. with 100 µg eOD or amph-eOD mixed with 375 µg SMNP adjuvant and boosted at 8, 16, and 24 weeks with the same formulations (red arrows in C and D). (A) Total IgM, IgG, and IgA secreting plasmablasts frequencies and (B) Percent antigen-specific IgM, IgG, and IgA secreting plasmablasts in peripheral blood were determined by ELISPOT; data are shown as a percent of total peripheral blood mononuclear cells (PBMCs). Statistical significance for (A) and (B) was determined using multiple unpaired t-tests. (C) Vaginal IgG and IgA titers were measured over time; (D) rectal IgG and IgA titers were measured over time. Statistical significance for (C) and (D) was determined using a two-way ANOVA comparing eOD and amph-eOD across all timepoints. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant. All data are presented as mean ± s.e.m.